A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein.

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Precisely Navigated Biobot Swarms of Bacteria Magnetospirillum magneticum for Water Decontamination.

Hybrid biological robots (biobots) prepared from living cells are at the forefront of micro-/nanomotor research due to their biocompatibility and versatility toward multiple applications. However, their precise maneuverability is essential for practical applications. Magnetotactic bacteria are hybrid biobots that produce magnetosome magnetite crystals, which are more stable than synthesized magnetite and can orient along the direction of earth's magnetic field. Herein, we used Magnetospirillum magneticum strain AMB-1 (M. magneticum AMB-1) for the effective removal of chlorpyrifos (an organophosphate pesticide) in various aqueous solutions by naturally binding with organic matter. Precision control of M. magneticum AMB-1 was achieved by applying a magnetic field. Under a programed clockwise magnetic field, M. magneticum AMB-1 exhibit swarm behavior and move in a circular direction. Consequently, we foresee that M. magneticum AMB-1 can be applied in various environments to remove and retrieve pollutants by directional control magnetic actuation.

Swarming Magnetic Microrobots for Pathogen Isolation from Milk.

Bovine mastitis produced by Staphylococcus aureus (S. aureus) causes major problems in milk production due to the staphylococcal enterotoxins produced by this bacterium. These enterotoxins are stable and cannot be eradicated easily by common hygienic procedures once they are formed in dairy products. Here, magnetic microrobots (MagRobots) are developed based on paramagnetic hybrid microstructures loaded with IgG from rabbit serum that can bind and isolate S. aureus from milk in a concentration of 3.42 104 CFU g-1 (allowable minimum level established by the United States Food and Drug Administration, FDA). Protein A, which is present on the cell wall of S. aureus, selectively binds IgG from rabbit serum and loads the bacteria onto the surface of the MagRobots. The selective isolation of S. aureus is confirmed using a mixed suspension of S. aureus and Escherichia coli (E. coli). Moreover, this fuel-free system based on magnetic robots does not affect the natural milk microbiota or add any toxic compound resulting from fuel catalysis. This system can be used to isolate and transport efficiently S. aureus and discriminate it from nontarget bacteria for subsequent identification. Finally, this system can be scaled up for industrial use in food production.

Is non-buffered DMEM solution a suitable medium for in vitro bioactivity tests?

Several laboratories had tested bioactivity of the materials in commercially available solution DMEM (Dulbecco's Modified Eagle's Medium) that is normally used for cultivation of cell cultures. The objective of this work was to find out whether it is possible to replace TRIS-buffered SBF currently used for bioactivity tests with the non-buffered DMEM solution. To understand the role of the organic part of the DMEM solution in the process of crystallization, we have prepared non-buffered solution simulating only its inorganic part (identified as I-solution). It was found that under static-dynamic test conditions calcite (CaCO3) and the amorphous phase of calcium phosphate (ACP) formed on the surface of the glass-ceramic (45S5 bioactive glass based) scaffold exposed to both solutions. Additionally, halite (NaCl) formed at the beginning of exposure to DMEM. Hydroxyapatite phase was not detected on the surface in either non-buffered solution. Organic components contained in the DMEM solution failed to prevent formation of crystalline phases. The present results indicate that it is not recommendable to use DMEM for bioactivity tests of glass-ceramic materials due to its low concentration of Ca2+ ions, high concentration of HCO3 - ions and the necessity to maintain sterile environment during the test.

Human UBL5 protein interacts with coilin and meets the Cajal bodies.

UBL5 protein, a structural homologue of ubiquitin, was shown to be involved in pre-mRNA splicing and transcription regulation in yeast and Caenorhabditis elegans, respectively. However, role of the UBL5 human orthologue is still elusive. In our study, we observed that endogenous human UBL5 that was localized in the nucleus, partially associates with Cajal bodies (CBs), nuclear domains where spliceosomal components are assembled. Simultaneous expression of exogenous UBL5 and coilin resulted in their nuclear colocalization in HeLa cells. The ability of UBL5 to interact with coilin was proved by GST pull-down assay using coilin that was either in vitro translated or extracted from HEK293T cells. Further, our results showed that the UBL5-coilin interaction was not influenced by coilin phosphorylation. These results suggest that UBL5 could be targeted to CBs via its interaction with coilin. Relation between human UBL5 protein and CBs is in the agreement with current observations about yeast orthologue Hub1 playing important role in alternative splicing.

SUMO-2/3 conjugates accumulating under heat shock or MG132 treatment result largely from new protein synthesis.

Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.

Using dot blot with immunochemical detection to evaluate global changes in SUMO-2/3 conjugation.

Small ubiquitin-related modifier-2/3 (SUMO-2/3) is a member of the ubiquitin-like (Ubl) protein family. Conjugation of SUMO-2/3 to target proteins is influenced by various stress conditions and chemical inhibitors. SUMO-2/3 conjugation may serve as a neuroprotective mechanism and may play a role in protein quality control. A method for screening global changes in SUMO-2/3 conjugation would facilitate further research of SUMO-2/3 cellular function. Here we show that dot blot with immunochemical detection allows evaluation of changes in global cellular SUMO-2/3 conjugation and offers an alternative to more laborious Western blot analysis. The method is based on a change of SUMO-2/3 signal intensity upon its conjugation. The dot blot analysis presented here is a time-saving method that enables screening of large numbers of samples and easy statistical evaluation of the results.